Antibodies to risperidone haptens and use thereof

ABSTRACT

Disclosed is an antibody which binds to risperidone, which can be used to detect risperidone in a sample such as in a competitive immunoassay method. The antibody can be used in a lateral flow assay device for point-of-care detection of risperidone, including multiplex detection of aripiprazole, olanzapine, quetiapine, and risperidone in a single lateral flow assay device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/971,475, filed Aug. 20, 2013, published Feb. 27, 2014, asUS20140057301, issued Sep. 5, 2017, as U.S. Pat. No. 9,751,953, andclaims the benefit of U.S. Provisional Application No. 61/691,615, filedAug. 21, 2012, the contents of each of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of immunoassays, and inparticular to antibodies that bind to risperidone which can be used inimmunoassays for detection of risperidone.

BACKGROUND

Schizophrenia is a chronic and debilitating psychiatric disorderaffecting approximately 0.45-1% of the world's population (van Os, J.;Kapur, S. “Schizophrenia” Lancet 2009, 374, 635-645). The principalgoals of treatment are to achieve sustained remission from psychoticsymptoms, reduce the risk and consequences of relapse, and improvepatient functioning and overall quality of life. While many patientswith schizophrenia are able to achieve symptom stability with theavailable antipsychotic medications, poor adherence to medication is acommon reason for relapse with daily administered oral medications.Several studies (Abdel-Baki, A.; Ouellet-Plamondon, C.; Malla, A.“Pharmacotherapy Challenges in Patients with First-Episode Psychosis”Journal of Affective Disorders 2012, 138, S3-S14) investigating theoutcomes of non-compliance have shown that patients with schizophreniawho do not take their medication as prescribed have higher rates ofrelapse, hospital admission and suicide as well as increased mortality.It is estimated that 40 to 75% of patients with schizophrenia havedifficulty adhering to a daily oral treatment regimen (Lieberman, J. A.;Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A.; Perkins,D. O.; Keefe, R. S. E.; Davis, S. M.; Davis, C. E.; Lebowitz, B. D.;Severe, J.; Hsiao, J. K. “Effectiveness of Antipyschotic Drugs inPatients with Chronic Schizophrenia” New England Journal of Medicine2005, 353(12), 1209-1223).

Therapeutic drug monitoring (TDM) is the quantification of serum orplasma concentrations of drugs, including anti-psychotic drugs, fortreatment monitoring and optimization. Such monitoring permits, forexample, the identification of patients that are not adhering to theirmedication regimen, that are not achieving therapeutic doses, that arenon-responsive at therapeutic doses, that have suboptimal tolerability,that have pharmacokinetic drug-drug interactions, or that have abnormalmetabolism resulting in inappropriate plasma concentrations.Considerable individual variability exists in the patient's ability toabsorb, distribute, metabolize, and excrete anti-psychotic drugs. Suchdifferences can be caused by concurrent disease, age, concomitantmedication or genetic peculiarities. Different drug formulations canalso influence the metabolism of anti-psychotic drugs. TDM permits doseoptimization for individual patients, improving therapeutic andfunctional outcomes. TDM further permits a prescribing clinician toensure compliance with prescribed dosages and achievement of effectiveserum concentrations.

To date, methods for determining the levels of serum or plasmaconcentrations of anti-psychotic drugs involve the use of liquidchromatography (LC) with UV or mass spectrometry detection, andradioimmunoassays (see, for example, Woestenborghs et al., 1990 “On theselectivity of some recently developed RIA's” in Methodological Surveysin Biochemistry and Analysis 20:241-246. Analysis of Drugs andMetabolites, Including Anti-infective Agents; Heykants et al., 1994 “ThePharmacokinetics of Risperidone in Humans: A Summary”, J Clin Psychiatry55/5, suppl:13-17; Huang et al., 1993 “Pharmacokinetics of the novelanti-psychotic agent risperidone and the prolactin response in healthysubjects”, Clin Pharmacol Ther 54:257-268). Radioimmunoassays detect oneor both of risperidone and paliperidone. Salamone et al. in U.S. Pat.No. 8,088,594 disclose a competitive immunoassay for risperidone usingantibodies that detect both risperidone and paliperidone but notpharmacologically inactive metabolites. The antibodies used in thecompetitive immunoassay are developed against a particular immunogen. IDLabs Inc. (London, Ontario, Canada) markets an ELISA for olanzapine,another anti-psychotic drug, which also utilizes a competitive format.The Instructions For Use indicate that the assay is designed forscreening purposes and intended for forensic or research use, and isspecifically not intended for therapeutic use. The Instructionsrecommend that all positive samples should be confirmed with gaschromatography/mass spectrometry (GC-MS), and indicate that the antibodyused detects olanzapine and clozapine (see ID Labs Inc., “InstructionsFor Use Data Sheet IDEL-F083”, Rev. Date Aug. 8, 2011). Some of thesemethods, namely HPLC and GC/MS, can be expensive and labor-intensive,and are generally only performed in large or specialty labs having theappropriate equipment.

A need exists for other methods for determining the levels ofanti-psychotic drugs, particularly methods that can be performed in aprescribing clinician's office (where the treatment for an individualpatient can be adjusted accordingly in a much more timely manner) and inother medical settings lacking LC or GC/MS equipment or requiring rapidtest results.

Risperidone is:

SUMMARY OF THE INVENTION

The present invention is directed to an isolated antibody or a bindingfragment thereof, which binds to risperidone and which: (i) is generatedin response to a conjugate of a compound of Formula I and an immunogeniccarrier; or (ii) competes for an epitope which is the same as an epitopebound by the antibody of (i).

Formula I:

wherein:R¹ is H, or OH;R² is O(CH₂)_(r)NH₂,

O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -heteroalkyl-;R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;r is 1, 2, 3, 4, or 5;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

Presently preferred embodiments of the antibody of the subject inventionare the antibodies designated 5-5 and 5-9 generated against thecompounds having Formula II and Formula III, and the antibody designated2A-5 generated against the compound having Formula IV. Other suitableimmunogens are the compounds having Formulas V and VI.

Formula II (Compound 4):

Formula III (Compound 5):

Formula IV (Compound 13):

Formula V (Compound 2):

Formula VI (Compound 12):

The antibodies of the subject invention can be provided in assay kitsand assay devices, with a presently preferred device being a lateralflow assay device which provides for point-of-care analysis.

The invention further provides a method of producing an antibody whichbinds to risperidone, the method comprising: (i) selecting a host cellfor antibody production; and (ii) inoculating the host with a conjugateof a compound of Formula I and an immunogenic carrier, wherein the hostproduces an antibody which binds to risperidone. Further provided is amethod of producing a hybridoma cell line capable of producing amonoclonal antibody which binds to risperidone. The method comprises:(i) selecting a host for antibody production; (ii) inoculating the hostwith a conjugate of a compound of Formula I and an immunogenic carrier;(iii) fusing a cell line from the inoculated host with a continuouslydividing cell to create a fused cell capable of producing a monoclonalantibody which binds to risperidone; and (iv) cloning the fused cell soas to obtain a hybridoma cell line.

The invention further provides a method of detecting risperidone in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and risperidone present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect risperidone in the sample.

Further provided is a competitive immunoassay method for detectingrisperidone in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and withrisperidone or a competitive binding partner of risperidone, wherein oneof the antibody and the risperidone or competitive binding partnerthereof is labeled with a detectable marker, and wherein samplerisperidone competes with the risperidone or competitive binding partnerthereof for binding to the antibody; and (ii) detecting the label so asto detect sample risperidone.

Further objects, features and advantages of the present invention willbe apparent to those skilled in the art from detailed consideration ofthe preferred embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show Competitive ELISA results generated with hybridoma5-9;

FIG. 3 shows Competitive ELISA results generated withrisperidone/paliperidone clone 2A5;

FIG. 4 shows the competitive immunoassay format used on a lateral flowassay device;

FIG. 5 shows a typical dose response curve generated withrisperidone/paliperidone clone 5-9;

FIG. 6 shows the chip design of a lateral flow assay device according tothe subject invention;

FIG. 7 shows a typical dose response curve for an aripiprazole positivecontrol generated with antibody 5C7 and a labeled aripiprazolecompetitive binding partner;

FIG. 8 shows a typical dose response curve for an olanzapine positivecontrol generated with antibody 4G9-1 and a labeled olanzapinecompetitive binding partner;

FIG. 9 shows a typical dose response curve for a quetiapine positivecontrol generated with antibody 11 and a labeled quetiapine competitivebinding partner;

FIG. 10 shows a typical dose response curve for a risperidone positivecontrol generated with antibody 5-9 and a labeled risperidonecompetitive binding partner;

FIG. 11 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence oflabeled aripiprazole competitive binding partner, with no dose responsecurve for olanzapine, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 12 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 13 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence of alabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 14 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence of alabeled competitive binding partner for each;

FIG. 15 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence ofa labeled aripiprazole competitive binding partner, with no doseresponse curve for olanzapine, quetiapine, or risperidone in thepresence of antibody and labeled competitive binding partner for each;

FIG. 16 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 17 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence oflabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 18 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence ofantibody and labeled competitive binding partner for each;

FIG. 19 shows a comparison of the aripiprazole dose response curvegenerated as a positive control to the aripiprazole dose response curvegenerated in the multiplex format;

FIG. 20 shows a comparison of the olanzapine dose response curvegenerated as a positive control to the olanzapine dose response curvegenerated in the multiplex format;

FIG. 21 shows a comparison of the quetiapine dose response curvegenerated as a positive control to the quetiapine dose response curvegenerated in the multiplex format; and

FIG. 22 shows a comparison of the risperidone dose response curvegenerated as a positive control to the risperidone dose response curvegenerated in the multiplex format.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides an isolated antibody which binds to risperidone.The invention further provides an assay kit and an assay devicecomprising the antibody. Also provided are methods of producing theantibody and of producing a hybridoma cell line capable of producing theantibody. Further provided is a method of detecting risperidone in asample, including a competitive immunoassay method.

In one embodiment, the present invention is directed to an isolatedantibody or a binding fragment thereof, which binds to risperidone andwhich: (i) is generated in response to a conjugate of a compound ofFormula I and an immunogenic carrier; or (ii) competes for an epitopewhich is the same as an epitope bound by the antibody of (i).

Formula I:

wherein:R¹ is H, or OH;R² is O(CH₂)_(r)NH₂,

O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;wherein:Z is selected from the group consisting of:—N(R⁴)—, —O—, —S—, -heteroalkyl-;R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;r is 1, 2, 3, 4, or 5;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H, or OH;

R² is O(CH₂)_(r)NH₂,

O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;wherein:Z is O;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;r is 1, 2, 3, 4, or 5;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H, or OH;

R² is O(CH₂)_(r)NH₂,

O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;wherein:Z is O(CH₂)_(r)NH;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 0, or 1;r is 1, 2, 3, 4, or 5;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H, or OH;

R² is O(CH₂)_(r)NH₂,O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;wherein:Z is O(CH₂)_(r)NH;Y is an organic spacer group;G is a functional linking group capable of binding to a carrier;p is 1;r is 2;m is 1, 2, 3, 4, or 5;n is 1, 2, 3, 4, or 5.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H, or OH;

R² is O(CH₂)_(r)NH₂,

O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H;wherein:r is 2;m is 1, 2, 3, or 4;n is 1, or 2.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula I and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i);wherein:

R¹ is H, or OH; and

R² is O(CH₂)_(r)NH₂, or

wherein r is 2;wherein m is 1.

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula VII and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula VIII and an immunogenic carrier; or (ii) competesfor an epitope which is the same as an epitope bound by the antibody of(i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula IX and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

In a preferred embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds torisperidone and which: (i) is generated in response to a conjugate of acompound of Formula X and an immunogenic carrier; or (ii) competes foran epitope which is the same as an epitope bound by the antibody of (i).

Preferably, the antibody of the subject invention is generated inresponse to a conjugate of a compound selected from the compounds of:Formula I, Formula VII, Formula VIII, Formula IX, and Formula X; and animmunogenic carrier.

Further details of the compounds described by the formulas above and theconjugates formed by the compounds and an immunogenic carrier areprovided in the section below entitled “Compounds, Conjugates andImmunogens”.

Further details of the antibodies of the subject invention are providedin the section below entitled “Antibodies”.

The subject invention further provides an assay kit comprising theantibody, as well as an assay device comprising the antibody.Preferably, the assay device is a lateral flow assay device. Furtherdetails of the assay kits and assay devices are provided below in thesection entitled “Assay Kits and Devices”.

The invention further provides a method of producing an antibody whichbinds to risperidone, the method comprising: (i) selecting a host cellfor antibody production; and (ii) inoculating the host with a conjugateof a compound of Formula I and an immunogenic carrier, wherein the hostproduces an antibody which binds to risperidone. In additionalembodiments, the conjugate used in the method can be a conjugate of acompound selected from the compounds of: Formula VII, Formula VIII,Formula IX, and Formula X; and an immunogenic carrier. Further detailson the production of the antibodies of the subject invention areprovided in the section below entitled “Antibodies”.

Further provided is a method of producing a hybridoma cell line capableof producing a monoclonal antibody which binds to risperidone. Themethod comprises: (i) selecting a host for antibody production; (ii)inoculating the host with a conjugate of a compound of Formula I and animmunogenic carrier; (iii) fusing a cell line from the inoculated hostwith a continuously dividing cell to create a fused cell capable ofproducing a monoclonal antibody which binds to risperidone; and (iv)cloning the fused cell so as to obtain a hybridoma cell line. Inadditional embodiments, the conjugate used in the method can be aconjugate of a compound selected from the compounds of: Formula VII,Formula VIII, Formula IX, and Formula X; and an immunogenic carrier.Further details of the production of hybridomas in accordance with thesubject invention are provided in the section below entitled“Antibodies”.

The invention further provides a method of detecting risperidone in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and risperidone present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect risperidone in the sample. Further details of the method ofdetecting risperidone in accordance with the subject invention areprovided in the section below entitled “Immunoassays”.

Further provided is a competitive immunoassay method for detectingrisperidone in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and withrisperidone or a competitive binding partner of risperidone, wherein oneof the antibody and the risperidone or competitive binding partnerthereof is labeled with a detectable marker, and wherein samplerisperidone competes with the risperidone or competitive binding partnerthereof for binding to the antibody; and (ii) detecting the label so asto detect sample risperidone. Further details of the competitiveimmunoassay method of detecting risperidone in accordance with thesubject invention are provided in the section below entitled“Immunoassays”.

In a preferred embodiment of the subject invention, the detection ofrisperidone is accompanied by the detection of one or more analytes inaddition to risperidone. Preferably the one or more analytes areanti-psychotic drugs other than risperidone, and more preferably theanti-psychotic drugs other than risperidone are selected from the groupconsisting of: aripiprazole, paliperidone, quetiapine, olanzapine, andmetabolites thereof.

As discussed above, the antibodies of the subject invention can be usedin assays to detect the presence and/or amount of the anti-psychoticdrug in patient samples. Such detection permits therapeutic drugmonitoring enabling all of the benefits thereof. Detection of levels ofanti-psychotic drugs may be useful for many purposes, each of whichrepresents another embodiment of the subject invention, including:determination of patient adherence or compliance with prescribedtherapy; use as a decision tool to determine whether a patient should beconverted from an oral anti-psychotic regimen to a long-actinginjectable anti-psychotic regimen; use as a decision tool to determineif the dose level or dosing interval of oral or injectableanti-psychotics should be increased or decreased to ensure attainment ormaintenance of efficacious or safe drug levels; use as an aid in theinitiation of anti-psychotic drug therapy by providing evidence of theattainment of minimum pK levels; use to determine bioequivalence ofanti-psychotic drug in multiple formulations or from multiple sources;use to assess the impact of polypharmacy and potential drug-druginteractions; and use as an indication that a patient should be excludedfrom or included in a clinical trial and as an aid in the subsequentmonitoring of adherence to clinical trial medication requirements.

Compounds, Conjugates and Immunogens

In relation to the compounds and conjugates and immunogens, thefollowing abbreviations are used: AMAS is N-(α-maleimidoacetoxy)succinimide ester; BTG is bovine thyroglobulin; Bu3N is tributylamine;DCC is dicyclohexylcarbodiimide; DCM is dichloromethane; DIEA isdiisopropylethylamine; DMF is N,N-dimethylformamide; EDTA isethylenediaminetetraaceticacid; KLH is keyhole limpet hemocyanin; SATAis N-succinimidyl S-acetylthioacetate; TEA is triethylamine; THF istetrahydrofuran; TFA is trifluoroacetic acid; Et3N is triethylamine;TBDMS is t-butyldimethylsilyl; DIC is diisopropylcarbodiimide; DMAP isN,N-dimethyl-4-aminopyridine; EDC is 1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride; NHS is N-hydroxysuccinimide; TFP isTetrafluorophenyl; PNP is p-nitrophenyl; TBTU isO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate;HOBT is N-Hydroxybenzotriazole; DEPBT is3-(diethoxyphosphoryloxy)-1,2,3-benzotrazi-4(3H)-one; BOP-Cl isBis(2-oxo-3-oxazolidinyl)phosphonic chloride; DTT is dithioerythritol.

The term “conjugate” refers to any substance formed from the joiningtogether of separate parts. Representative conjugates include thoseformed by the joining together of a small molecule, such as thecompounds of Formula I, and a large molecule, such as a carrier or apolyamine polymer, particularly a protein. In the conjugate the smallmolecule may be joined at one or more active sites on the largemolecule.

The term “hapten” refers to a partial or incomplete antigen. A hapten isa protein-free substance, which is not capable of stimulating antibodyformation, but which does react with antibodies. The antibodies areformed by coupling a hapten to a high molecular weight immunogeniccarrier, and then injecting this coupled product, i.e., an immunogen,into a human or animal subject.

The term “immunogen” refers to a substance capable of eliciting,producing, or generating an immune response in an organism.

An “immunogenic carrier,” as used herein, is an immunogenic substance,commonly a protein, that can join at one or more positions with haptens,thereby enabling the production of antibodies that can bind with thesehaptens. Examples of immunogenic carrier substances include, but are notlimited to, proteins, glycoproteins, complex polyamino-polysaccharides,particles, and nucleic acids that are recognized as foreign and therebyelicit an immunologic response from the host. Thepolyamino-polysaccharides may be prepared from polysaccharides using anyof the conventional means known for this preparation.

Various protein types may be employed as immunogenic carriers, includingwithout limitation, albumins, serum proteins, lipoproteins, etc.Illustrative proteins include bovine serum albumin, keyhole limpethemocyanin, egg ovalbumin, bovine thyroglobulin, fraction V human serumalbumin, rabbit albumin, pumpkin seed globulin, diphtheria toxoid,tetanus toxoid, botilinus toxin, succinylated proteins, and syntheticpoly(aminoacids) such as polylysine.

Immunogenic carriers can also include poly amino-polysaccharides, whichare a high molecular weight polymers built up by repeated condensationsof monosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide also contains poly(amino acid) residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “analogue” of a chemical compound refers to a chemical compoundthat contains a chain of carbon atoms and the same particular functionalgroups as a reference compound, but the carbon chain of the analogue islonger or shorter than that of the reference compound.

A “label,” “detector molecule,” “reporter” or “detectable marker” is anymolecule which produces, or can be induced to produce, a detectablesignal. The label can be conjugated to an analyte, immunogen, antibody,or to another molecule such as a receptor or a molecule that can bind toa receptor such as a ligand, particularly a hapten or antibody. A labelcan be attached directly or indirectly by means of a linking or bridgingmoiety. Non-limiting examples of labels include radioactive isotopes(e.g., ¹²⁵I), enzymes (e.g. 3-galactosidase, peroxidase), enzymefragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts,fluorophores (e.g., rhodamine, fluorescein isothiocyanate or FITC, orDylight 649), dyes, chemiluminescers and luminescers (e.g., dioxetanes,luciferin), or sensitizers.

As used herein, a “spacer” refers to a portion of a chemical structurewhich connects two or more substructures such as haptens, carriers,immunogens, labels or binding partners through a functional linkinggroup. These spacer groups are composed of the atoms typically presentand assembled in ways typically found in organic compounds and so may bereferred to as “organic spacing groups”. The chemical building blocksused to assemble the spacers will be described hereinafter in thisapplication. Among the preferred spacers are straight or branched,saturated or unsaturated carbon chains. These carbon chains may alsoinclude one or more heteroatoms within the chain, one or moreheteroatoms replacing one or more hydrogens of any carbon atom in thechain, or at the termini of the chains. By “heteroatoms” is meant atomsother than carbon which are chosen from the group consisting of oxygen,nitrogen, phosphorous and sulfur, wherein the nitrogen, phosphorous andsulfur atoms may exist in any oxidation state and may have carbon orother heteroatoms bonded to them. The spacer may also include cyclic oraromatic groups as part of the chain or as a substitution on one of theatoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. Preferred chain lengths are between 1 to 20 atoms.

A “functional linking group” refers to a reactive group that is presenton a hapten and may be used to provide an available reactive sitethrough which the hapten portion may be coupled to another moietythrough formation of a covalent chemical bond to produce a conjugate ofa hapten with another moiety (such as a label or carrier). The haptenmay be linked in this way to a moiety such as biotin to form acompetitive binding partner.

Spacer groups may be used to link the hapten to the carrier. Spacers ofdifferent lengths allow one to attach the hapten with differingdistances from the carrier for presentation to the immune system of theanimal or human being immunized for optimization of the antibodyformation process. Attachment to different positions in the haptenmolecule allows the opportunity to present specific sites on the haptento the immune system to influence antibody recognition. The spacer maycontain hydrophilic solubilizing groups to make the hapten derivativemore soluble in aqueous media. Examples of hydrophilic solubilizinggroups include but are not limited to polyoxyalkyloxy groups, forexample, polyethylene glycol chains; hydroxyl, carboxylate and sulfonategroups.

The term “nucleophilic group” or “nucleophile” refers to a species thatdonates an electron-pair to form a chemical bond in a reaction. The term“electrophilic group” or “electrophile” refers to a species that acceptsan electron-pair from a nucleophile to form a chemical bond in areaction.

The term “substituted” refers to substitution of an atom or group ofatoms in place of a hydrogen atom on a carbon atom in any position onthe parent molecule. Non limiting examples of substituents includehalogen atoms, amino, hydroxy, carboxy, alkyl, aryl, heteroalkyl,heteroaryl, cyano, alkoxy, nitro, aldehyde and ketone groups.

The term “alkyl” refers to saturated or unsaturated linear and branchedchain radicals of up to 12 carbon atoms, unless otherwise indicated, andis specifically intended to include radicals having any degree or levelof saturation. Alkyl includes, but is not limited to, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, hexyl, isohexyl, heptyl, octyl, 2,2,4-trimethylpentyl, nonyl,decyl, undecyl and dodecyl.

The term “cycloalkyl” refers to a saturated or partially unsaturatedmonocyclic or bicyclic hydrocarbon ring radical composed of from 3 to 10carbon atoms. Alkyl substituents may optionally be present on the ring.Examples include cyclopropyl, 1,1-dimethyl cyclobutyl,1,2,3-trimethylcyclopentyl, cyclohexyl and cyclohexenyl.

The term “heteroalkyl” refers to an alkyl group that includes one ormore heteroatoms within the chain, one or more heteroatoms replacing oneor more hydrogens of any carbon atom in the chain, or at termini of thechains.

The term “aminoalkyl” refers to at least one primary or secondary aminogroup bonded to any carbon atom along an alkyl chain.

The term “alkoxy” refers to straight or branched chain radicals of up to12 carbon atoms, unless otherwise indicated, bonded to an oxygen atom.Examples include but are not limited to methoxy, ethoxy, propoxy,isopropoxy and butoxy.

The term “alkoxyalkyl” refers to at least one alkoxy group bonded to anycarbon atom along an alkyl chain.

The term “thioalkyl” refers to at least one sulfur group bonded to anycarbon atom along an alkyl chain. The sulfur group may be at anyoxidation state and includes sulfoxides, sulfones and sulfates.

The term “carboxylate group” includes carboxylic acids and alkyl,cycloalkyl, aryl or aralkyl carboxylate esters.

The term “alkylcarbonyl” refers to a group that has a carbonyl groupbonded to any carbon atom along an alkyl chain.

The term “heteroaryl” refers to 5- to 7-membered mono- or 8- to10-membered bicyclic aromatic ring radicals, any ring of which mayconsist of from one to four heteroatoms selected from N, O or S wherethe nitrogen and sulfur atoms can exist in any allowed oxidation state.Examples include benzimidazolyl, benzothiazolyl, benzothienyl,benzoxazolyl, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl,pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl,thiazolyl and thienyl.

The term “aryl” refers to monocyclic or bicyclic aromatic ring radicalscontaining from 6 to 12 carbons in the ring. Alkyl substituents mayoptionally be present on the ring. Examples include phenyl, biphenyl andnapththalene.

The term “aralkyl” refers to a C₁₋₆ alkyl group containing an arylsubstituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl.

The term “acyl” refers to the group —C(O)R_(a), where R_(a) is hydrogen,alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl and heteroaryl. An“acylating agent” adds the —C(O)R_(a) group to a molecule.

The term “sulfonyl” refers to the group —S(O)₂R_(b), where R_(b) ishydrogen, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aryl, aralkyl andheteroaryl. A “sulfonylating agent” adds the —S(o)₂R_(a) group to amolecule.

Spacers bearing reactive functional linking groups for the attachment ofhaptens to carrier moieties may be prepared by a wide variety ofmethods. The spacer may be formed using a molecule that isdifferentially functionalized or activated with groups at either end toallow selective sequential reaction with the hapten and the carrier, butthe same reactive moiety may also be used at both ends. The groupsselected for reaction with the hapten and the functional linking groupto be bound to the carrier are determined by the type of functionalityon the hapten and the carrier that the hapten is to be bonded with.Spacers and methods of attachment to haptens and carriers include butare not limited to those described by Brinkley, M., A., BioconjugateChem. 1992, 3:2-13, Hermanson, Greg T., Bioconjugate Techniques,Academic Press, London, Amsterdam, Burlington, Mass., USA, 2008 andThermo Scientific Pierce Crosslinking Technical Handbook; available fordownload or hard copy request from Thermo Scientific 3747 N Meridian Rd,Rockford, Ill. USA 61101, ph 800-874-3723 or at World Wide Web internetsite “piercent.com” and references within. Many differentially activatedmolecules for formation of spacer groups are commercially available fromvendors, for example Thermo Scientific.

For haptens bearing an amino group, modes of attachment of the spacer tothe hapten include reaction of the amine on the hapten with a spacerbuilding block bearing an acyl halide or active ester. “Active esters”are defined as esters that undergo reaction with a nucleophilic group,for example an amino group, under mild conditions to form a stablelinkage. A stable linkage is defined as one that remains intact underconditions of further use, for example subsequent synthetic steps, useas an immunogen, or in a biochemical assay. A preferred example of astable linkage is an amide bond. Active esters and methods of formationare described by Benoiton, N. L., in Houben-Weyl, Methods of OrganicChemistry, Thieme Stuttgart, New York, vol E22 section 3.2:443 andBenoiton, N. L., Chemistry of Peptide Synthesis, Taylor and Francis,N.Y., 2006. Preferred active esters include p-nitrophenyl ester (PNP),N-hydroxysuccinimide ester (NHS) and tetrafluorophenyl ester (TFP). Acylhalides may be prepared by many methods known to one skilled in the artfor example, reaction of the carboxylic acid with thionyl chloride oroxalyl chloride, see: Fieser, L. F. and Fieser, M. Reagents for OrganicSynthesis, John Wiley and Sons, N.Y., 1967 and references within. Thesemay be converted to other active esters such as p-nitrophenyl esters(PNP) which may also be used in active bi-functional spacers asdescribed by Wu et. al, Organic Letters, 2004, 6 (24):4407.N-hydroxysuccinimide (NHS) esters may be prepared by reaction ofN,N-disuccinimidyl carbonate (CAS 74124-79-1) with the carboxylic acidof a compound in the presence of an organic base such as triethylamineor diisopropylethylamine in an aprotic solvent under anhydrousconditions as described in Example 35 of WO2012012595 or by usingN-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) or otherdehydrating agent, under anhydrous conditions. Tetrafluorophenyl esters(TFP) may be prepared by reaction of carboxylic acids with2,3,5,6-tetrafluorophenyltrifluoroacetate in the presence of an organicbase such as triethylamine or diisopropylethylamine in an aproticsolvent under anhydrous conditions as reported by Wilbur, et. al,Bioconjugate Chem., 2004, 15(1):203. One skilled in the art willrecognize that spacers shown in Table 1, among others, can be obtainedusing known methods and attached to amino-bearing haptens utilizingroutine optimization of reaction conditions. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 1

Reasonable values for m and n are between 1 and 10

Direct coupling of the amine on the hapten and a carboxylic acidfunctionality on the spacer building block in the presence of a couplingagent may also be used as a mode of attachment. Preferred reagents arethose typically used in peptide synthesis. Peptide coupling reagentsinclude but are not limited toO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU, CAS #125700-67-6), see: Pruhs, S., Org. Process. Res. Dev. 2006,10:441; N-Hydroxybenzotriazole (HOBT, CAS #2592-95-2) with acarbodiimide dehydrating agent, for example N—N-dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC), or1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), see:Konig W., Geiger, R. Chem. Ber., 1970, 103 (3):788;3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one (DEPBT, CAS#165534-43-0), see: Liu, H. et. al., Chinese Chemical Letters, 2002,13(7):601; Bis(2-oxo-3-oxazolidinyl)phosphonic chloride; (BOP-Cl, CAS#68641-49-6), see: Diago-Meseguer, J et. al. Synthesis, 1980, 7:547-51and others described in detail by Benoiton in Chemistry of PeptideSynthesis, CRC Press, Boca Raton, Fla., 2005, Chapter 2, and thetechnical bulletin provided by Advanced Automated Peptide ProteinTechnologies (aapptec), 6309 Shepardsville Rd., Louisville Ky. 40228, ph888 692 9111; www.aapptec.com, and references within. These methodscreate a stable amide linkage attaching the hapten to the spacer.Examples of spacers that can be obtained using known methods andattached to amino-bearing haptens utilizing routine optimization ofreaction conditions employing the methods described and cited above areshown, but not limited to those in Table 2. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 2

reasonable range for n is between 1-10

Spacers may also be constructed in a step-wise fashion by sequentialattachment of appropriate chemical groups to the hapten including thestep of forming the functional linking group that is capable of bindingto the carrier. See illustrative examples under General ReactionSchemes.

Additionally, when the hapten has a nucleophilic group, for example athiol group, an amino group or a hydroxyl group which will become thepoint of attachment of the spacer, the spacer may also be constructed byalkylation of the thiol, amine or hydroxyl group. Any alkyl group thatis appropriately substituted with a moiety capable of undergoing asubstitution reaction, for example, an alkyl halide, or sulfonic acidester such as p-Toluenesulfonate, may be used to attach the spacer. Manyexamples of alkylation reactions are known to one skilled in the art andspecific examples may be found in the general chemical literature andoptimized through routine experimentation. A discussion of alkylationreactions with many references can be found in Chapter 10 of March'sAdvanced Organic Chemistry, Smith, M. B., and March, J., John Wiley &sons, Inc. N.Y., 2001. Other linkages may also be employed such asreaction of the nucleophilic moiety, for example an amine, on the haptenwith an isocyanate to form a urea or reaction with an isothiocyanate toform a thiourea linkage, see: Li, Z., et. al., Phosphorus, Sulfur andSilicon and the Related Elements, 2003, 178(2):293-297. Spacers may beattached to haptens bearing hydroxyl groups via reaction with isocyanategroups to form carbamate or urethane linkages. The spacer may bedifferentially activated with the isocyanate functional group on one endand a functional linking group capable of reacting with the carrier,see: Annunziato, M. E., Patel, U.S., Ranade, M. and Palumbo, P. S.,Bioconjugate Chem., 1993, 4:212-218.

For haptens bearing a carboxylic acid group, modes of attachment of aspacer portion to the hapten include activation of the carboxylic acidgroup as an acyl halide or active ester, examples of which are shown inTable 3, preparation of which are described previously, followed byreaction with an amino (—NH₂—), hydrazino (—NH—NH₂—), hydrazido(—C(O)—NH—NH₂—) or hydroxyl group (—OH) on the spacer portion to form anamide, hydrazide, diacylhydrazine or ester linkage, or direct couplingof the carboxylic acid group with an amino group on the spacer portionor directly on the carrier with a peptide coupling reagent and/orcarbodiimide dehydrating reagent, described previously, examples ofwhich are shown in Tables 4 and 5. Procedures found in references citedpreviously for formation of activated esters and use of peptide couplingagents may be employed for attachment of carboxylic acid-bearing haptensto spacer building blocks and protein carriers with available aminogroups utilizing routine optimization of reaction conditions.

TABLE 3

TABLE 4

TABLE 5

Other electrophilic groups may be present on the hapten to attach thespacer, for example, a sulfonyl halide

or electrophilic phosphorous group, for example:

See: Malachowski, William P., Coward, James K., Journal of OrganicChemistry, 1994, 59 (25):7616or:

R_(c) is alkyl, cycloalkyl, aryl, substituted aryl, aralkyl.

See: Aliouane, L., et. al, Tetrahedron Letters, 2011, 52(28):8681.

Haptens that bear aldehyde or ketone groups may be attached to spacersusing methods including but not limited to reaction with a hydrazidegroup H₂N—NH—C(O)— on the spacer to form an acylhydrazone, see: Chamow,S. M., Kogan, T. P., Peers, D. H., Hastings, R. C., Byrn, R. A. andAskenaszi, A., J. Biol. Chem., 1992, 267(22): 15916. Examples ofbifunctional hydrazide spacer groups that allow attachment to a thiolgroup on the carrier are shown in Table 6.

TABLE 6

Haptens may also contain thiol groups which may be reacted with thecarrier provided that the carrier has been modified to provide a groupthat may react with the thiol. Carrier groups may be modified by methodsincluding but not limited to attachment of a group containing amaleimide functional group by reaction of an amino group on the carrierwith N-Succinimidyl maleimidoacetate, (AMAS, CAS #55750-61-3),Succinimidyl iodoacetate (CAS #151199-81-4), or any of the bifunctionalspacer groups shown in Table 1 to introduce a group which may undergo areaction resulting in attachment of the hapten to the carrier.

The functional linking group capable of forming a bond with the carriermay be any group capable of forming a stable linkage and may be reactiveto a number of different groups on the carrier. The functional linkinggroup may preferably react with an amino group, a carboxylic acid groupor a thiol group on the carrier, or derivative thereof. Non-limitingexamples of the functional linking group are a carboxylic acid group,acyl halide, active ester (as defined previously), isocyanate,isothiocyanate, alkyl halide, amino group, thiol group, maleimide group,acrylate group (H₂C═CH—C(O)—) or vinyl sulfone group H₂C═CH—SO₂—) See:Park, J. W., et. al., Bioconjugate Chem., 2012, 23(3): 350. Thefunctional linking group may be present as part of a differentiallyactivated spacer building block that may be reacted stepwise with thehapten and the resulting hapten derivative may then be reacted with thecarrier. Alternatively, the hapten may be derivatized with a spacer thatbears a precursor group that may be transformed into the functionallinking group by a subsequent reaction. When the functional linkinggroup on the spacer is an amine or a carboxylic acid group, the couplingreaction with the carboxylic acid group or amine on the carrier may becarried out directly through the use of peptide coupling reagentsaccording to procedures in the references cited above for thesereagents.

Particular disulfide groups, for example, pyridyldisulfides, may be usedas the functional linking group on the spacer which may undergo exchangewith a thiol group on the carrier to from a mixed disulfide linkage,see: Ghetie, V., et al., Bioconjugate Chem., 1990, 1:24-31. Thesespacers may be attached by reaction of the amine-bearing hapten with anactive ester which is attached to a spacer bearing the pyridyldisulfidegroup, examples of which include but are not limited to those shown inTable 7.

TABLE 7

Most often the carrier is a protein and the ε-amino groups of the lysineresidues may be used for attachment, either directly by reaction with anamine-reactive functional linking group or after derivitization with athiol-containing group, including N-Succinimidyl S-Acetylthioacetate,(SATA, CAS 76931-93-6), or an analogue thereof, followed by cleavage ofthe actetate group with hydroxylamine to expose the thiol group forreaction with the functional linking group on the hapten. Thiol groupsmay also be introduced into the carrier by reduction of disulfide bondswithin protein carriers with mild reducing reagents including but notlimited to 2-mercaptoethylamine, see: Bilah, M., et. al.,Bioelectrochemistry, 2010, 80(1):49, phosphine reagents, see: Kirley, T.L., Analytical Biochemistry, 1989, 180(2):231 or dithioerythritol (DTT,CAS 3483-12-3) Cleland, W., Biochemistry, 1964, 3:480-482.

General Reaction Schemes

Compounds useful for producing antibodies according to the subjectinvention can be synthesized in accordance with the general syntheticmethods described below. Compounds of Formula (I) can be prepared bymethods known to those who are skilled in the art. The followingreaction schemes are only meant to represent examples of the inventionand are in no way meant to be a limit of the invention.

Attachment of a spacer to the parent ring structure of risperidone maybe accomplished through use of the silyl-protected starting compoundshown in Scheme 1, the preparation of which is described in Example 1.Alkylation with an N-protected haloalkyl derivative is also described inExample 1. N-protected haloalkyl derivatives of varying chain lengthsare commercially available or may be made by standard organic reactionsknown to one skilled in the art. Preferred values for r are between 1and 5. Deprotection as described in Example 1 may provide the aminocompound which may be further elaborated to attach additional spaceratoms or may be linked directly to the carrier. Derivatives of the aminocompound which lack the hydroxyl group in final product may be made asdescribed in Example 3.

Alkylation of risperidone may also be accomplished using a thiol, forexample, 3-mercaptomethylpropionate, using the method of Wang, J., L.,et. al., Bioorganic and Med. Chem. Letters, 2010, 20:7159, using K₂CO₃in DMF followed by hydrolysis with NaOH in aqueous THF, as shown inScheme 2, to provide an analogue of the hapten that bears a thioalkyllinkage terminating in a carboxy group that may be attached directly toa carrier or further elaborated to extend the spacer portion. Alkylationwith an amine may be carried out also as shown in Scheme 2, according tothe method used to make intermediate 535 in US20110245224. Versions ofthe aminoalkyl or thioalkyl analogues of Scheme 2 in which R1 is eitherOH or H may be made through routine optimization of the chemicalprocedures taught in the aforementioned references by a skilled chemist.

The phenolic hydroxyl group of the starting compound shown in Scheme 3,wherein R₁ is either H or a silyl-protected alcohol, may be the site ofintroduction of a group that bears a functional linking group forattachment to a carrier. The phenolic compound may be reacted directlywith succinic anhydride as shown in Scheme 3 and described inUS20060251592 to provide a carboxy-bearing intermediate, or may bereacted with an isocyanate bifunctional spacer, as shown in Scheme 4,according to the Annunziato reference provided elsewhere in thisdisclosure to provide a hapten that bears a thiol-reactive functionallinker. Deprotection as described in subsequent examples is requiredwhen R₁ is a silyl-protected alcohol.

Scheme 5 illustrates how haptens with spacers which terminate in analkyl amine group, such as Examples 1 and 2, may be furtherfunctionalized with a maleimide group. The maleimide may be introducedby any method known in the art. For example, reaction withN-maleoyl-substituted alkyl amino acid in a solvent such asdichlorormethane and coupling reagents such as diisopropylethylamine anddiethyl cyanophosphonate gives the maleimide functionalized spacer onthe hapten. Reaction of the risperidone derived amine withalkyl-maleimide functionalizing group, such as 2,5-dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate, in a solvent such asDMF, in the presence of a base, such as tributylamine, at roomtemperature for about an hour, generates haptens with a maleimidefunctionalized spacer.

Haptens with spacers which terminate in an alkyl amine group may beelaborated by reaction with a cyclic anhydride compound, such assuccinic anhydride or glutaric anhydride, as shown in Scheme 6. Thereaction may be carried out in a solvent such as THF, at roomtemperature, overnight.

Maleimide functionalized haptens may be conjugated to proteins accordingto the method shown in Scheme 7. Activation of protein lysine residuesby acylation of the epsilon-nitrogen with N-succinimidylS-acetylthioacetate (SATA), followed by subsequent hydrolysis of theS-acetyl group with hydroxylamine produces a nucleophilic sulfhydrylgroup. Conjugation of the sulfhydryl activated protein with themaleimide derivatized hapten (prepared as described in general scheme 5)proceeds via a Michael addition reaction. Suitable proteins are known tothose skilled in the art and include keyhole limpet hemocyanin, bovinethyroglobulin, and ovalbumin.

Carboxylic acid functionalized haptens may be conjugated to proteinsaccording to the method shown in Scheme 8. Reaction withN-hydroxysuccinimide and a suitable coupling agent, such asdicyclohexylcarbodiimide (DCC), and a base, such as tributylamine, in asolvent such as DMF at a temperature of about 20° C., for about 18hours, activates the carboxylic acid with the N-hydroxysuccinimideleaving group. The activated spacer and hapten may then be conjugated toa protein in a solvent such as pH 7.5 phosphate buffer, at about 20° C.for about 2.5 hours. Suitable proteins are known to those skilled in theart and include keyhole limpet hemocyanin, bovine thyroglobulin, andovalbumin.

Antibodies

The present invention is directed to an isolated antibody or a bindingfragment thereof, which binds to risperidone and which: (i) is generatedin response to a conjugate of a compound of Formula I and an immunogeniccarrier; or (ii) competes for an epitope which is the same as an epitopebound by the antibody of (i). The term “antibody” refers to a specificprotein capable of binding an antigen or portion thereof (in accordancewith this invention, capable of binding to an anti-psychotic drug ormetabolite thereof). An antibody is produced in response to an immunogenwhich may have been introduced into a host, e.g., an animal or a human,by injection. The generic term “antibody” includes polyclonalantibodies, monoclonal antibodies, and antibody fragments.

“Antibody” or “antigen-binding antibody fragment” refers to an intactantibody, or a fragment thereof, that competes with the intact antibodyfor binding. Generally speaking, an antibody or antigen-binding antibodyfragment, is said to specifically bind an antigen when the dissociationconstant is less than or equal to 1 μM, preferably less than or equal to100 nM and most preferably less than or equal to 10 nM. Binding can bemeasured by methods know to those skilled in the art, an example beingthe use of a BIAcore™ instrument.

Antibody fragments comprise a portion of an intact antibody, preferablythe antigen binding or variable region of the intact antibody. Bindingfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. An antibody other than a“bispecific” or “bifunctional” antibody is understood to have each ofits binding sites identical.

As used herein, “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Two antibodies are said to “bind thesame epitope” if one antibody is shown to compete with the secondantibody in a competitive binding assay, by any of the methods wellknown to those skilled in the art (such as the BIAcore™ method referredto above). In reference to a hapten (such as risperidone or otheranti-psychotic drug), an antibody can be generated against thenon-antigenic hapten molecule by conjugating the hapten to animmunogenic carrier. An antibody is then generated which recognizes an“epitope” defined by the hapten.

“Isolated” when used in the context of an antibody means altered “by thehand of man” from any natural state; i.e., that, if it occurs in nature,it has been changed or removed from its original environment, or both.For example, a naturally occurring antibody naturally present in aliving animal in its natural state is not “isolated”, but the sameantibody separated from the coexisting materials of its natural state is“isolated”, as the term is employed herein. Antibodies may occur in acomposition, such as an immunoassay reagent, which are not naturallyoccurring compositions, and therein remain isolated antibodies withinthe meaning of that term as it is employed herein.

“Cross-reactivity” refers to the reaction of an antibody with an antigenthat was not used to induce that antibody.

Preferably, the antibody of the subject invention will bind to the drugand any desired pharmacologically active metabolites. By altering thelocation of the attachment of the immunogenic carrier to the compoundsof the invention, selectivity and cross-reactivity with metabolites canbe engineered into the antibodies. For risperidone, cross-reactivitywith risperidone metabolites such as 9-hydroxyrisperidone (paliperidone,which is also administered as an anti-psychotic drug),7-hydroxyrisperidone, and N-dealkylrisperidone may or may not bedesirable. An antibody that cross-reacts with risperidone andpaliperidone may be desirable, which does not react with7-hydroxyrisperidone or N-dealkylrisperidone, thus detecting risperidoneand its major pharmacologically active metabolite. Alternatively, it maybe desirable to detect the pharmacologically active metabolites,risperidone and paliperidone, separately, while still not detecting theinactive metabolites, 7-hydroxyrisperidone and N-dealkylrisperidone.Antibodies may be generated that detect multiple ones of these drugsand/or metabolites, or antibodies may be generated that detect eachseparately (thus defining the antibody “specific binding” properties).An antibody specifically binds one or more compounds when its binding ofthe one or more compounds is equimolar or substantially equimolar.

Methods of producing such antibodies comprise inoculating a host withthe conjugate described herein. Suitable hosts include, but are notlimited to, mice, rats, hamsters, guinea pigs, rabbits, chickens,donkeys, horses, monkeys, chimpanzees, orangutans, gorillas, humans, andany species capable of mounting a mature immune response. Theimmunization procedures are well established in the art and are setforth in numerous treatises and publications including “The ImmunoassayHandbook”, 2nd Edition, edited by David Wild (Nature Publishing Group,2000) and the references cited therein.

Preferably, an immunogen embodying features of the present invention isadministered to a host subject, e.g., an animal or human, in combinationwith an adjuvant. Suitable adjuvants include, but are not limited to,Freund's adjuvant, powdered aluminum hydroxide (alum), aluminumhydroxide together with Bordetella pertussis, and monophosphoryl lipidA-synthetic trehalose dicorynomycolate (MPL-TDM).

Typically, an immunogen or a combination of an immunogen and an adjuvantis injected into a mammalian host by one or multiple subcutaneous orintraperitoneal injections. Preferably, the immunization program iscarried out over at least one week, and more preferably, over two ormore weeks. Polyclonal antibodies produced in this manner can beisolated and purified utilizing methods well know in the art.

Monoclonal antibodies can be produced by the well-established hybridomamethods of Kohler and Milstein, e.g., Nature 256:495-497 (1975).Hybridoma methods typically involve immunizing a host or lymphocytesfrom a host, harvesting the monoclonal antibody secreting or having thepotential to secrete lymphocytes, fusing the lymphocytes to immortalizedcells, and selecting cells that secrete the desired monoclonal antibody.

A host can be immunized to elicit lymphocytes that produce or arecapable of producing antibodies specific for an immunogen.Alternatively, the lymphocytes can be immunized in vitro. If human cellsare desired, peripheral blood lymphocytes can be used, although spleencells or lymphocytes from other mammalian sources are preferred.

The lymphocytes can be fused with an immortalized cell line to formhybridoma cells, a process which can be facilitated by the use of afusing agent, e.g., polyethylene glycol. By way of illustration, mutantrodent, bovine, or human myeloma cells immortalized by transformationcan be used. Substantially pure populations of hybridoma cells, asopposed to unfused immortalized cells, are preferred. Thus, followingfusion, the cells can be grown in a suitable medium that inhibits thegrowh or survival of unfused, immortalized cells, for example, by usingmutant myeloma cells that lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT). In such an instance, hypoxanthine,aminopterin, and thymidine can be added to the medium (HAT medium) toprevent the growth of HGPRT-deficient cells while permitting hybridomasto grow.

Preferably, immortalized cells fuse efficiently, can be isolated frommixed populations by selection in a medium such as HAT, and supportstable and high-level expression of antibody following fusion. Preferredimmortalized cell lines include myeloma cell lines available from theAmerican Type Culture Collection, Manassas, Va.

Because hybridoma cells typically secrete antibody extracellularly, theculture media can be assayed for the presence of monoclonal antibodiesspecific for the anti-psychotic drug. Immunoprecipitation of in vitrobinding assays, for example, radiioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA), can be used to measure the bindingspecificity of monoclonal antibodies.

Monoclonal antibody-secreting hybridoma cells can be isolated as singleclones by limiting dilution procedures and sub-cultured. Suitableculture media include, but are not limited to, Dulbecco's ModifiedEagle's Medium, RPMI-1640, and polypeptide-free, polypeptide-reduced, orserum-free media, e.g., Ultra DOMA PF or HL-1, available fromBiowhittaker, Walkersville, Md. Alternatively, the hybridoma cells canbe grown in vivo as ascites.

Monoclonal antibodies can be isolated and/or purified from a culturemedium or ascites fluid by conventional immunoglobulin (Ig) purificationprocedures including, but not limited to, polypeptide A-SEPHAROSE,hydroxylapatite chromatography, gel electrophoresis, dialysis, ammoniumsulfate precipitation, and affinity chromatography.

Monoclonal antibodies can also be produced by recombinant methods suchas are described in U.S. Pat. No. 4,166,452. DNA encoding monoclonalantibodies can be isolated and sequenced using conventional procedures,e.g., using oligonucleotide probes that specifically bind to murineheavy and light antibody chain genes, preferably to probe DNA isolatedfrom monoclonal antibody hybridoma cells lines secreting antibodiesspecific for anti-psychotic drugs.

Antibody fragments which contain specific binding sites for theanti-psychotic drug may also be generated. Such fragments include, butare not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al., Science 256:1270-1281 (1989)). Fab, Fvand ScFv antibody fragments can all be expressed in and secreted fromEscherichia coli, allowing for the production of large amounts of thesefragments. Alternatively, Fab′-SH fragments can be directly recoveredfrom E. coli and chemically coupled to form F(ab′)₂ fragments (Carter etal., BioTechnology 10:163-167 (1992)). Other techniques for theproduction of antibody fragments are known to those skilled in the art.Single chain Fv fragments (scFv) are also envisioned (see U.S. Pat. Nos.5,761,894 and 5,587,458). Fv and sFv fragments are the only species withintact combining sites that are devoid of constant regions; thus, theyare likely to show reduced non-specific binding. The antibody fragmentmay also be a “linear antibody” e.g., as described in U.S. Pat. No.5,642,870, for example. Such linear antibody fragments may bemonospecific or bispecific.

Assay Kits and Devices

An assay kit (also referred to as a reagent kit) can also be providedcomprising an antibody as described above. A representative reagent kitmay comprise an antibody that binds to the anti-psychotic drug,risperidone, a complex comprising an analog of an anti-psychotic drug ora derivative thereof coupled to a labeling moiety, and may optionallyalso comprise one or more calibrators comprising a known amount of ananti-psychotic drug or a related standard.

The phrase “assay kit” refers to an assembly of materials and reagentsthat is used in performing an assay. The reagents can be provided inpackaged combination in the same or in separate containers, depending ontheir cross-reactivities and stabilities, and in liquid or inlyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. An assay kit embodying features of the presentinvention comprises antibodies which bind risperidone. The kit mayfurther comprise competitive binding partners of risperidone andcalibration and control materials.

The phrase “calibration and control material” refers to any standard orreference material containing a known amount of an analyte. A samplesuspected of containing an analyte and the corresponding calibrationmaterial are assayed under similar conditions. The concentration ofanalyte is calculated by comparing the results obtained for the unknownspecimen with the results obtained for the standard. This is commonlydone by constructing a calibration curve.

Antibodies embodying features of the present invention can be includedin a kit, container, pack, or dispenser together with instructions fortheir utilization. When the antibodies are supplied in a kit, thedifferent components of the immunoassay may be packaged in separatecontainers and admixed prior to use. Such packaging of the componentsseparately may permit long-term storage without substantiallydiminishing the functioning of the active components. Furthermore,reagents can be packaged under inert environments, e.g., under apositive pressure of nitrogen gas, argon gas, or the like, which isespecially preferred for reagents that are sensitive to air and/ormoisture.

Reagents included in kits embodying features of the present inventioncan be supplied in all manner of containers such that the activities ofthe different components are substantially preserved while thecomponents themselves are not substantially adsorbed or altered by thematerials of the container. Suitable containers include, but are notlimited to, ampules, bottles, test tubes, vials, flasks, syringes,envelopes, e.g., foil-lined, and the like. The containers may becomprised of any suitable material including, but not limited to, glass,organic polymers, e.g., polycarbonate, polystyrene, polyethylene, etc.,ceramic, metal, e.g., aluminum, metal alloys, e.g., steel, cork, and thelike. In addition, the containers may comprise one or more sterileaccess ports, e.g., for access via a needle, such as may be provided bya septum. Preferred materials for septa include rubber andpolytetrafluoroethylene of the type sold under the trade name TEFLON byDuPont (Wilmington, Del.). In addition, the containers may comprise twoor more compartments separated by partitions or membranes that can beremoved to allow mixing of the components.

Reagent kits embodying features of the present invention may also besupplied with instructional materials. Instructions may be printed,e.g., on paper and/or supplied in an electronically-readable medium.Alternatively, instructions may be provided by directing a user to aninternet website, e.g., specified by the manufacturer or distributor ofthe kit and/or via electronic mail.

The antibody may also be provided as part of an assay device. Such assaydevices include lateral flow assay devices. A common type of disposablelateral flow assay device includes a zone or area for receiving theliquid sample, a conjugate zone, and a reaction zone. These assaydevices are commonly known as lateral flow test strips. They employ aporous material, e.g., nitrocellulose, defining a path for fluid flowcapable of supporting capillary flow. Examples include those shown inU.S. Pat. Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660 all ofwhich are incorporated herein by reference in their entireties.

Another type of assay device is a non-porous assay device havingprojections to induce capillary flow. Examples of such assay devicesinclude the open lateral flow device as disclosed in PCT InternationalPublication Nos. WO 2003/103835, WO 2005/089082, WO 2005/118139, and WO2006/137785, all of which are incorporated herein by reference in theirentireties.

In a non-porous assay device, the assay device generally has at leastone sample addition zone, at least one conjugate zone, at least onereaction zone, and at least one wicking zone. The zones form a flow pathby which sample flows from the sample addition zone to the wicking zone.Also included are capture elements, such as antibodies, in the reactionzone, capable of binding to the analyte, optionally deposited on thedevice (such as by coating); and a labeled conjugate material alsocapable of participating in reactions that will enable determination ofthe concentration of the analyte, deposited on the device in theconjugate zone, wherein the labeled conjugate material carries a labelfor detection in the reaction zone. The conjugate material is dissolvedas the sample flows through the conjugate zone forming a conjugate plumeof dissolved labeled conjugate material and sample that flows downstreamto the reaction zone. As the conjugate plume flows into the reactionzone, the conjugated material will be captured by the capture elementssuch as via a complex of conjugated material and analyte (as in a“sandwich” assay) or directly (as in a “competitive” assay). Unbounddissolved conjugate material will be swept past the reaction zone intothe at least one wicking zone. Such devices can include projections ormicropillars in the flow path.

An instrument such as that disclosed in US Patent Publication Nos.US20060289787A1 and US 20070231883A1, and U.S. Pat. Nos. 7,416,700 and6,139,800, all of which are incorporated herein by reference in theirentireties, is able to detect the bound conjugated material in thereaction zone. Common labels include fluorescent dyes that can bedetected by instruments which excite the fluorescent dyes andincorporate a detector capable of detecting the fluorescent dyes.

Immunoassays

The antibodies thus produced can be used in immunoassays torecognize/bind to the anti-psychotic drug, thereby detecting thepresence and/or amount of the drug in a patient sample. Preferably, theassay format is a competitive immunoassay format. Such an assay formatand other assays are described, among other places, in Hampton et al.(Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

The term “analyte” refers to any substance or group of substances, thepresence or amount of which is to be determined. Representativeanti-psychotic drug analytes include, but are not limited to,risperidone, paliperidone, olanzapine, aripiprazole, and quetiapine.

The term “competitive binding partner” refers to a substance or group ofsubstances, such as may be employed in a competitive immunoassay, whichbehave similarly to an analyte with respect to binding affinity to anantibody. Representative competitive binding partners include, but arenot limited to, anti-psychotic drug derivatives and the like.

The term “detecting” when used with an analyte refers to anyquantitative, semi-quantitative, or qualitative method as well as to allother methods for determining an analyte in general, and ananti-psychotic drug in particular. For example, a method that merelydetects the presence or absence of an anti-psychotic drug in a samplelies within the scope of the present invention, as do methods thatprovide data as to the amount or concentration of the anti-psychoticdrug in the sample. The terms “detecting”, “determining”, “identifying”,and the like are used synonymously herein, and all lie within the scopeof the present invention.

A preferred embodiment of the subject invention is a competitiveimmunoassay wherein antibodies which bind the anti-psychotic drug, orthe drug or competitive binding partner thereof, are attached to a solidsupport (such as the reaction zone in a lateral flow assay device) andlabeled drug or competitive binding partner thereof, or labeledantibody, respectively, and a sample derived from the host are passedover the solid support and the amount of label detected attached to thesolid support can be correlated to a quantity of drug in the sample.

Any sample that is suspected of containing an analyte, e.g., ananti-psychotic drug, can be analyzed in accordance with the methods ofthe presently preferred embodiments. The sample can be pretreated ifdesired and can be prepared in any convenient medium that does notinterfere with the assay. Preferably, the sample comprises an aqueousmedium such as a body fluid from a host, most preferably plasma orserum.

It is to be understood that all manner of immunoassays employingantibodies are contemplated for use in accordance with the presentlypreferred embodiments, including assays in which antibodies are bound tosolid phases and assays in which antibodies are in liquid media. Methodsof immunoassays that can be used to detect analytes using antibodiesembodying features of the present invention include, but are not limitedto, competitive (reagent limited) assays wherein labeled analyte(analyte analog) and analyte in a sample compete for antibodies andsingle-site immunometric assays wherein the antibody is labeled; and thelike.

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Habor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Copending applications entitled “Haptens of Aripiprazole” (U.S.Provisional Patent Appl. No. 61/691,450, filed Aug. 21, 2012, and US20140163206, filed Aug. 20, 2013), “Haptens of Olanzapine” (U.S.Provisional Patent Appl. No. 61/691,454, filed Aug. 21, 2012, and US20140213766, filed Aug. 20, 2013), “Haptens of Paliperidone” (U.S.Provisional Patent Appl. No. 61/691,459, filed Aug. 21, 2012 and US2014/0213767, filed Aug. 20, 2013), “Haptens of Quetiapine” (U.S.Provisional Patent Appl. No. 61/691,462, filed Aug. 21, 2012, and US20140221616, filed Aug. 20, 2013), “Haptens of Risperidone andPaliperidone” (U.S. Provisional Patent Appl. No. 61/691,469, filed Aug.21, 2012, and US 20140155585, Aug. 20, 2013), “Antibodies toAripiprazole Haptens and Use Thereof” (U.S. Provisional Patent Appl. No.61/691,544, filed Aug. 21, 2012, and US 20140057299, filed Aug. 20,2013), “Antibodies to Olanzapine Haptens and Use Thereof” (U.S.Provisional Patent Appl. No. 61/691,572, filed Aug. 21, 2012, US20140057303, filed Aug. 20, 2013), “Antibodies to Paliperidone Haptensand Use Thereof” (U.S. Provisional Patent Appl. No. 61/691,634, filedAug. 21, 2012, and US 20140057297, filed Aug. 20, 2013), “Antibodies toQuetiapine Haptens and Use Thereof” (U.S. Provisional Patent Appl. No.61/691,598, filed Aug. 21, 2012, and US 20140057305, filed Aug. 20,2013), “Antibodies to Aripiprazole and Use Thereof” (U.S. ProvisionalPatent Appl. No. 61/691,522, filed Aug. 21, 2012, and US 20140057300,filed Aug. 20, 2013), “Antibodies to Olanzapine and Use Thereof” (U.S.Provisional Patent Appl. No. 61/691,645, filed Aug. 21, 2012, and US20140057304, filed Aug. 20, 2013), “Antibodies to Paliperidone and UseThereof” (U.S. Provisional Patent Appl. No. 61/691,692, filed Aug. 21,2012, and US 20140057298, filed Aug. 20, 2013), “Antibodies toQuetiapine and Use Thereof” (U.S. Provisional Patent Appl. No.61/691,659, filed Aug. 21, 2012, and US 20140057306, filed Aug. 20,2013), “Antibodies to Risperidone and Use Thereof” (U.S. ProvisionalPatent Appl. No. 61/691,675, filed Aug. 21, 2012, and US 20140057302,filed Aug. 20, 2013), and “Antibodies to Risperidone and Use Thereof”(U.S. Provisional Patent Appl. No. 61/790,880, filed Mar. 15, 2013, andUS 20140057302, filed Aug. 20, 2013) are all incorporated herein byreference in their entireties.

EXAMPLE 1 Step A9-((tert-butyldimethylsilypoxy)-3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

A solution of3-(2-chloroethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one(1.0 g, 4.12 mmol) in DMF (5 mL) was treated with 1H-imidazole (701.24mg, 64.66 mmol), followed by a solution of t-butyldimethylchlorosilane(683.12 mg, 4.53 mmol) in DMF (1 mL). After stirring for 18 h at roomtemperature, the solvents were removed under vacuum and the residue wastaken up in dichloromethane/water (10 mL/10 mL) with addition of aspatula of potassium carbonate. The aqueous layer was extracted withdichloromethane (three times 10 mL). The combined organic fractions weredried over Na₂SO₄, filtered, and the solvent was removed under vacuum.The crude mixture was used without further purification in the nextstep. (ESI-MS (M+1) 357).

Step B9-((tert-butyldimethylsilypoxy)-3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

A solution of9-((tert-butyldimethylsilyl)oxy)-3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,prepared as described in the previous step, (0.5 g, 1.40 mmol) inmethanol (25 mL) and diisopropylethylamine (732.83 μL, 4.20 mmol) wastreated with 3-(piperidin-4-yl)benzo[d]isoxazol-6-ol hydrochloride salt(374.62 mg, 1.47 mmol), and the reaction mixture was stirred for 17 h at60° C. under argon. Diisopropylethylamine (732.83 μL, 4.20 mmol) wasadded and the mixture was stirred additionally for 4 h at 60° C. Thereaction mixture was evaporated under vacuum and the residue was takenup in water (25 mL), extracted with chloroform (3×25 mL). The combinedorganic layers were dried over MgSO₄, filtered, and the solvent wasremoved under vacuum. The residue was purified by silica gelchromatography (elution with dichloromethane/methanol (98/2) to give thetitle compound (ESI-MS (M+1) 539).

Step C tert-butyl(2-((3-(1-(2-(9-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)carbamate

A solution of9-((tert-butyldimethylsilyl)oxy)-3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,prepared as described in the previous step, (50 mg, 0.093 mmol) inacetone (0.5 mL) and DMF (0.5 mL) was treated with potassium carbonate(33.3 mg, 0.24 mmol) and N-Boc-2-bromoaminoethane (27 mg, 0.12 mmol),and the reaction mixture was stirred for 17 h at 60° C. under argon. Thereaction mixture was evaporated at 40° C. under reduced pressure anddissolved in water (10 mL) and extracted with dichloromethane (3×10 mL).The organic layers were combined, dried over Na₂SO₄, filtered, and thesolvent was evaporated yield the crude title compound. (ESI-MS (M+1)682).

Step D3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

A solution of tert-butyl(2-((3-(1-(2-(9-((tert-butyldimethylsilyl)oxy)-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)carbamate,prepared as described in the previous step, (70 mg, 0.103 mmol) inHCl/isopropanol (10 mL, 5 N) was stirred for 1 h at 60° C. The reactionmixture was evaporated at 40° C. under reduced pressure and dissolvedcarefully in aqueous saturated sodium bicarbonate solution (5 mL) andextracted with dichloromethane (3×10 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and evaporated under reduced pressureat 40° C. The aqueous layer still contained product, which was recoveredby evaporating the aqueous layer to dryness at 40° C. under reducedpressure. The resulting residue from the aqueous layer was re-dissolvedin water and brought over a conditioned Waters Oasis SPE (6 cc) columnand afterwards eluted with methanol. The methanol elution fraction wascombined with the residue of the dichloromethane extraction andevaporated to dryness at 40° C. under reduced pressure to yield thetitle compound along with a side product (ESI-MS (M+1) 468; side productbeing 5%9-hydroxy-3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one(M+1) 425). The mixture was used in the next step without additionalpurification.

EXAMPLE 22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide

To a solution of3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-9-hydroxy-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,prepared as described in Example 1, (4.0 mg, 8.5 μmoles) in 215 μL ofDMF and 4.3 μL of tributylamine was added 214 μL of a DMF solution ofN-(α-maleimidoacetoxy) succinimide ester (AMAS, 10 mg/mL, 2.1 mg, 8.5μmoles). The resulting solution was allowed to stir for 60 minutes at20° C., then used as such in conjugation reaction with thiol-activatedprotein.

EXAMPLE 3 Step A3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

A solution of3-(2-chloroethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one(14.4 g, 0.05 mmol), 3-(piperidin-4-yl)benzo[d]isoxazol-6-ol (14.0 g,0.05 mmol), sodium carbonate (16.0 g, 0.15 mmol) and potassium iodide(spatula point) in DMF (150 mL) was stirred for 5 h at 80° C. Themixture was allowed to cool down to room temperature and water wasadded. The precipitate was removed by filtration, and the filtrate wasextracted with chloroform (3×100 mL). The combined organic layers weredried over Na₂SO₄, filtered, and concentrated. The residue wascrystallized with isopropyl alcohol (70 mL), filtered, and washed withisopropanol/diisopropyl ether 50/50 mixture (10 mL). The residue wasdried overnight at 100° C. yielding the title compound and was usedwithout further purification in the next step.

Step B3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one

A solution of3-(2-(4-(6-hydroxybenzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,prepared as described in the previous step, (6.6 g, 0.015 mmol) in DMF(50 mL) and acetone (50 mL) was treated with potassium carbonate (3.0 g,0.03 mmol) and ethyl (2-bromoethyl)carbamate (2.4 g, 0.015 mmol). Afterstirring overnight at 60° C., the reaction mixture was poured in water(150 mL), extracted with chloroform (3×100 mL). The combined organiclayers were dried over Na₂SO₄, filtered, concentrated, and purified bysilica gel chromatography (elution with dichloromethane/methanol(90/10). The combined fractions were treated with HBr (150 mL, 48%) andheated to reflux for 30 min. The mixture was allowed to cool down toroom temperature and made basic with ammonium hydroxide (28% NH₃ in H₂O)and extracted with chloroform (3×100 mL). The combined organic layerswere dried over Na₂SO₄, filtered, concentrated, and purified by silicagel chromatography (gradient elution with dichloromethane/methanol(90/10 to 50/50) resulting in a solid which was dissolved in isopropanol(50 mL) and treated with isopropanol/HCl. The precipitate was removed byfiltration and washed with iPrOH/diisopropyl ether (50/50, 3×20 mL). Theprecipitate was dried under vacuum to yield the title compound ESI-MS(M+1) 452. ¹H NMR (360 MHz, DMSO-d6) δ ppm 1.76-1.85 (m, 1H) 1.87-1.96(m, 1H) 2.19 (d, J=12.81 Hz, 1H) 2.37-2.48 (m, 4H) 2.98-3.10 (m, 3H)3.10-3.28 (m, 5H) 3.37-3.46 (m, 3H) 3.72 (d, J=11.34 Hz, 3H) 3.79-3.85(m, 2H) 4.31 (t, J=4.94 Hz, 1H) 7.05 (dd, J=8.78, 1.83 Hz, 1H) 7.35-7.39(m, 1H) 8.08 (d, J=8.78 Hz, 1H).

EXAMPLE 42-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide

To a solution of3-(2-(4-(6-(2-aminoethoxy)benzo[d]isoxazol-3-yl)piperidin-1-yl)ethyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one,prepared as described in Example 3, (3.4 mg, 7.58 μmoles) in 185 μL ofDMF and 3.7 μL of tributylamine was added 190 μL of a DMF solution ofN-(α-maleimidoacetoxy) succinimide ester (AMAS, 10 mg/mL, 1.9 mg, 7.58μmoles). The resulting solution was allowed to stir for 90 minutes at20° C., then used as such in conjugation reaction with thiol-activatedprotein.

EXAMPLE 52-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide—KeyholeLimpet Hemocyanin—Conjugate

Step A

To a 4.22 mL solution of keyhole limpet hemocyanin (KLH, 18.0 mg, 0.18μmoles) in 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4 wasadded 83.2 μL of a DMF solution of N-succinimidyl-S-acetylthioacetate(SATA, 25 mg/mL, 2.1 mg, 9.0 μmoles). The resulting solution wasincubated at 20° C. for 1 hour on a roller mixer. The reaction waspurified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46 Msodium chloride, 5 mM EDTA, at pH 6.0.

Step B

To 9.37 mL of KLH-SATA solution, prepared as described in Step A, (17.1mg, 0.171 μmoles) was added 937 μL of 2.5 M hydroxylamine, 50 mM EDTA,at pH 7.0. The resulting solution was incubated at 20° C. for 40 minuteson a roller mixer. The reaction was used as such in conjugation reactionwith maleimide-activated hapten.

Step C

To an aliquot of the resulting KLH-SH solution, prepared as described inStep B, (3.4 mL, 0.058 μmoles) was added an aliquot of2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamidesolution, prepared as described in Example 2, (282.8 μL, 5.0 μmoles).The resulting cloudy mixture was incubated for 3 hours at 20° C. on aroller mixer. The reaction was filtered through a 0.2 μm syringe filterthen purified on a Sephadex G-25 column using 100 mM phosphate buffer,0.46M sodium chloride, at pH 7.4.

EXAMPLE 62-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide—BovineThyroglobulin—Conjugate

Step A

To 1.0 mL of a solution of bovine thyroglobulin (BTG, 9.3 mg, 0.014μmoles) in 100 mM phosphate buffer at pH 7.5 was added 132 μL of a DMFsolution of N-succinimidyl-S-acetylthioacetate (SATA, 25 mg/mL, 3.3 mg,14.1 μmoles). The resulting solution was incubated at 20° C. for 1 houron a roller mixer. The reaction was purified on a Sephadex G-25 columnusing 100 mM phosphate buffer, 5 mM EDTA, at pH 6.0.

Step B

To 2.11 mL of BTG-SATA solution, prepared as described in Step A, (7.4mg, 0.011 μmoles) was added 211 μL of 2.5 M hydroxylamine, 50 mM EDTA,at pH 7.0. The resulting solution was incubated at 20° C. for 60 minuteson a roller mixer. The reaction was used as such in conjugation reactionwith maleimide-activated hapten.

Step C

To an aliquot of the resulting BTG-SH solution, prepared as described inStep B, (2.3 mL, 0.011 μmoles) was added an aliquot of2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(9-hydroxy-2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamidesolution, prepared as described in Example 2, (280.4 μL, 5.5 μmoles).The resulting cloudy mixture was incubated for 2.5 hours at 20° C. on aroller mixer. The reaction was filtered through a 0.2 μm syringe filterthen purified on a Sephadex G-25 column using 100 mM phosphate buffer,0.14M sodium chloride, at pH 7.4.

EXAMPLE 72-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide—KeyholeLimpet Hemocyanin—Conjugate

To an aliquot of KLH-SH solution, prepared as described in Example 5Step B, (1.5 mL, 0.025 μmoles) was added an aliquot of2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamidesolution, prepared as described in Example 4, (113 μL, 2.26 μmoles). Theresulting cloudy mixture was incubated for 2.5 hours at 20° C. on aroller mixer. The reaction was filtered through a 0.2 μm syringe filterthen purified on a Sephadex G-25 column using 100 mM phosphate buffer,and 0.46M sodium chloride, at pH 7.4.

EXAMPLE 82-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamide—BovineThyroglobulin—Conjugate

To an aliquot of BTG-SH solution, prepared as described in Example 6Step B, (0.63 mL, 0.0033 μmoles) was added an aliquot of of2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((3-(1-(2-(2-methyl-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-3-yl)ethyl)piperidin-4-yl)benzo[d]isoxazol-6-yl)oxy)ethyl)acetamidesolution, prepared as described in Example 4 (80 μL, 1.6 μmoles). Theresulting cloudy mixture was incubated for 2.5 hours at 20° C. on aroller mixer. The reaction was filtered through a 0.2 μm syringe filterthen purified on a Sephadex G-25 column using 100 mM phosphate buffer,and 0.14M sodium chloride, at pH 7.4.

EXAMPLE 9 Competitive Immunoassays for Risperidone/Paliperidone andMultiplex Competitive Immunoassay for Aripiprazole, Olanzapine,Quetiapine, and Risperidone/Paliperidone

Following a series of immunizations with paliperidone/risperidoneimmunogens, mouse tail bleeds were tested for reactivity using an ELISA.Hybridoma supernatants were also tested, and the ELISA data shown inTables 8 and 9 below shows reactivity of several hybridomas (fusionpartner was NSO cells). As shown in Table 9, reactivity of hybridomas2A5 and 5G11 was seen.

TABLE 8 Dilution 1 2 3 4 5 6 7 8 9 10 11 12 400 1 5 14 39 41 47 58 62 6772 76 Blank Ag = 1200 Bt- 3600 Com- 10800 pound#1 400 1 5 14 39 41 47 5862 67 72 76 1200 3600 10800 Dilutio 1 2 3 4 5 6 7 8 9 10 11 12 4003.2562 3.2897 3.3148 3.6038 0.6857 3.3976 1.3444 2.8639 0.5676 3.59932.5144 0.0143 Ag = 1200 1.3591 1.4605 1.521 2.3063 0.1476 1.9245 0.28411.0387 0.1158 2.6921 0.8711 0.0142 Bt- 3600 0.3745 0.4617 0.3733 0.76130.038 0.6163 0.0689 0.2742 0.0304 0.9549 0.2236 0.0115 Cmpd# 108000.0918 0.1149 0.0908 0.1919 0.0156 0.1834 0.0199 0.0639 0.013 0.27660.056 0.0099 1 400 3.1217 3.1103 3.1532 3.633 0.6089 3.5705 1.10672.4001 0.4963 3.4172 2.2432 0.0095 1200 1.2607 1.4817 1.3412 2.14110.1327 1.9831 0.2691 0.961 0.1027 2.5321 0.7418 0.0098 3600 0.32810.4159 0.3819 0.7373 0.0361 0.593 0.0723 0.292 0.0284 0.8426 0.20240.0079 10800 0.0879 0.1127 0.0929 0.1949 0.0156 0.189 0.0229 0.07220.0141 0.2393 0.052 0.0086

TABLE 9 Plate 1 Dilution 1 2 3 neat Blank 1C4 6 E6 neat 2A5 7A7 neat2G10 Empty neat 3B7 neat 4D8 neat 5A12 neat 5G11 neat 6C1 neat 0.00720.038 0.0309 neat 0.0077 3.9563 0.1163 neat 0.0069 0.0093 0.0086 neat0.0076 0.0753 0.0108 neat 0.0114 0.1139 0.0084 neat 0.009 0.0193 0.0123neat 0.0087 0.2503 0.0085 neat 0.0092 0.086 0.0121

After clones were identified via ELISA reactivity, competition ELISAswere run to approximate affinity and cross-reactivity with similarcompounds. FIGS. 1 and 2 show the ELISA cross-reactivity results fromhybridoma subclone 5_9. Data shows reactivity to risperidone, as well asits metabolites paliperidone and 7-hydroxyrisperidone.

Supernatants were also tested by competition ELISA to determine if thesignals were specific to either risperidone or paliperidone. FIG. 3shows the results from hybridoma subclone 2A5. Data shows reactivity toboth risperidone and paliperidone.

FIG. 4 shows the competitive immunoassay format used on a lateral flowassay device in which the capture antibody, risperidone/paliperidoneclone 5-9, was deposited on a chip along with a detection conjugateconsisting of risperidone conjugated to a fluorophore. In thiscompetitive format as show in FIG. 4, a low level of analyte(paliperidone) results in high signal, whereas a high level of analyte(paliperidone) results in low signal. The amount of paliperidone in thesample can be calculated from the loss of fluorescence compared to acontrol sample with no drug present. A typical dose response curvegenerated with risperidone/paliperidone clone 5-9 is shown in FIG. 5.

FIG. 6 shows the chip design of a lateral flow assay device according toone embodiment of the subject invention. The device includes a zone orarea for receiving the sample, a conjugate zone (which contains desiredlabeled competitive binding partner(s)), and a reaction zone (eightareas within the reaction zone are indicated; each area can contain aseparate desired antibody). Sample flows from the sample zone throughthe conjugate zone and to the reaction zone.

FIGS. 7-10 show typical dose response curves for an aripiprazolepositive control (sample containing aripiprazole) generated withantibody 5C7 deposited in reaction zone 2 and a labeled aripiprazolecompetitive binding partner in the conjugate zone (FIG. 7), anolanzapine positive control (sample containing olanzapine) generatedwith antibody 4G9-1 deposited in reaction zone 4 and a labeledolanzapine competitive binding partner in the conjugate zone (FIG. 8), aquetiapine positive control (sample containing quetiapine) generatedwith antibody 11 deposited in reaction zone 6 and a labeled quetiapinecompetitive binding partner in the conjugate zone (FIG. 9), and arisperidone positive control (sample containing risperidone) generatedwith antibody 5-9 deposited in reaction zone 8 and a labeled risperidonecompetitive binding partner in the conjugate zone (FIG. 10). The labeledcompetitive binding partners in the conjugate zone compete with thedrugs present in the samples for binding to the antibodies. The amountof label is detected and is an indication of the amount of drug presentin the sample (the amount of signal being inversely proportional to theamount of drug in the sample—see FIG. 4).

In order to confirm that conjugates of labeled competitive bindingpartners do not bind to antibodies deposited in the reaction zones,negative controls were conducted by using samples containing no drugs.Referring to Table 10, a sample containing no aripiprazole is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled olanzapine, labeled quetiapine, andlabeled risperidone, but no labeled aripiprazole) and to the reactionzone. The reaction zone again contains aripiprazole antibody (5C7) inreaction zone 2. Table 10 below shows the results, confirming that thereis no dose response and the olanzapine, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the aripiprazole antibody.

TABLE 10 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) ReactionRead Peak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground ARIP-MM1 OLAN, QUET, RISP ARIP 2 0.77 1.56 3.99 ARIP-MM1OLAN, QUET, RISP 4 −0.02 0.06 4.14 ARIP-MM1 OLAN, QUET, RISP 6 0.09 0.104.29 ARIP-MM1 OLAN, QUET, RISP 8 0.13 0.12 4.61 Other Conjugates do notbind to Aripiprazole

Referring to Table 11, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled quetiapine, andlabeled risperidone, but no labeled olanzapine) and to the reactionzone. The reaction zone again contains olanzapine antibody (4G9-1) inreaction zone 4. Table 11 below shows the results, confirming that thereis no dose response and the aripiprazole, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the olanzapine antibody.

TABLE 11 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Conc.) Reaction ReadPeak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground OLAN-MM1 ARIP, QUET, RISP 2 −0.03 0.05 4.38 OLAN-MM1 ARIP,QUET, RISP OLAN 4 0.74 1.10 4.56 OLAN-MM1 ARIP, QUET, RISP 6 0.06 0.094.79 OLAN-MM1 ARIP, QUET, RISP 8 0.11 0.13 5.17 Other Conjugates do notbind to Olanzapine

Referring to Table 12, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled olanzapine, andlabeled risperidone, but no labeled quetiapine) and to the reactionzone. The reaction zone again contains quetiapine antibody (11) inreaction zone 6. Table 12 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the quetiapine antibody.

TABLE 12 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Reaction ReadPeak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground QUET-MM1 ARIP, OLAN, RISP 2 −0.01 0.07 3.85 QUET-MM1 ARIP,OLAN, RISP 4 0.01 0.12 4.01 QUET-MM1 ARIP, OLAN, RISP QUET 6 0.03 0.084.24 QUET-MM1 ARIP, OLAN, RISP 8 0.04 0.07 4.56 Other Conjugates do notbind to Quetiapine

Referring to Table 13, a sample containing no risperidone is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled aripiprazole, labeled olanzapine, andlabeled quetiapine, but no labeled risperidone) and to the reactionzone. The reaction zone again contains risperidone antibody (5-9) inreaction zone 8. Table 13 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and quetiapineconjugates that move by capillary action through the reaction zone donot bind to the risperidone antibody.

TABLE 13 Risperidone-Clone 5-9-Math Model 1 (0 ng/mL Conc.) ReactionRead Peak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground RISP-MM1 ARIP, OLAN, QUET 2 0.02 0.11 7.43 RISP-MM1 ARIP,OLAN, QUET 4 0.05 0.14 7.73 RISP-MM1 ARIP, OLAN, QUET 6 0.20 0.19 8.11RISP-MM1 ARIP, OLAN, QUET RISP 8 1.97 3.23 8.85 Other Conjugates do notbind to Risperidone

In order to confirm that conjugates of labeled competitive bindingpartners bind only to their respective antibodies deposited in thereaction zones, additional negative controls were conducted by againusing samples containing no drugs. Referring to Table 14, a samplecontaining no aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (this time containinglabeled aripiprazole) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2, as well asolanzapine antibody (4G9-1) in reaction zone 4, quetiapine antibody (11)in reaction zone 6, and risperidone antibody (5-9) in reaction zone 8.Table 14 below shows the results, confirming that there is no doseresponse except to the aripiprazole antibody 5C7 (in reaction zone 2).

TABLE 14 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) ReactionRead Peak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground ARIP-MM1 ARIP, OLAN, QUET, RISP ARIP 2 60.34 97.53 5.44ARIP-MM1 ARIP, OLAN, QUET, RISP 4 2.86 3.91 11.66 ARIP-MM1 ARIP, OLAN,QUET, RISP 6 1.12 1.23 11.03 ARIP-MM1 ARIP, OLAN, QUET, RISP 8 3.14 4.1912.94 Only the Aripiprazole Reaction Zone is binding

Referring to Table 15, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled olanzapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 15 below shows the results, confirmingthat there is no dose response except to the olanzapine antibody 4G9-1(in reaction zone 4).

TABLE 15 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Reaction Read Peak MeanPeak Mean Mean Assay-MM Conj Zone Position Area Height BackgroundOLAN-MM1 ARIP, OLAN, QUET, RISP 2 0.0 0.0 4.86 OLAN-MM1 ARIP, OLAN,QUET, RISP OLAN 4 34.2 51.8 5.39 OLAN-MM1 ARIP, OLAN, QUET, RISP 6 0.20.3 5.39 OLAN-MM1 ARIP, OLAN, QUET, RISP 8 0.1 0.1 5.59 Only theOlanzapine Reaction Zone is binding

Referring to Table 16, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled quetiapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 16 below shows the results, confirmingthat there is no dose response except to the quetiapine antibody 11 (inreaction zone 6).

TABLE 16 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Reaction ReadPeak Mean Peak Mean Mean Assay-MM Conj Zone Position Area HeightBackground QUET-MM1 ARIP, OLAN, QUET, RISP 2 0.13 0.41 10.02 QUET-MM1ARIP, OLAN, QUET, RISP 4 0.08 0.23 10.47 QUET-MM1 ARIP, OLAN, QUET, RISPQUET 6 140.35 181.33 7.91 QUET-MM1 ARIP, OLAN, QUET, RISP 8 1.58 2.6111.53 Only the Quetiapine Reaction Zone is binding

Referring to Table 17, a sample containing no risperidone is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled risperidone) and to the reactionzone. The reaction zone again contains aripiprazole antibody (5C7) inreaction zone 2, as well as olanzapine antibody (4G9-1) in reaction zone4, quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 17 below shows the results, confirmingthat there is no dose response except to the risperidone antibody 5-9(in reaction zone 8).

TABLE 17 Risperidone-Clone 5-9-Math Model (0 ng/mL Reaction Read PeakMean Peak Mean Mean Assay-MM Conj Zone Position Area Height BackgroundRISP-MM1 ARIP, OLAN, QUET, RISP 2 1.03 1.51 9.07 RISP-MM1 ARIP, OLAN,QUET, RISP 4 0.65 0.91 9.60 RISP-MM1 ARIP, OLAN, QUET, RISP 6 2.61 6.3910.48 RISP-MM1 ARIP, OLAN, QUET, RISP RISP 8 55.98 100.91 11.58 Only theRisperidone Reaction Zone is binding

The results shown above confirm that conjugates of labeled competitivebinding partners bind only to their respective antibodies in thereaction zone.

FIGS. 11-14 show typical dose response curves in specific antibodyreaction zones, and proof of dose response low/high concentration foreach specific assay in the presence of other conjugates. In FIG. 11, asample containing aripiprazole is deposited in the sample zone and movesby capillary action through the conjugate zone (this time containinglabeled aripiprazole, labeled olanzapine, labeled quetiapine, andlabeled risperidone) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2. A typical doseresponse curve was generated as is shown in FIG. 11 only foraripiprazole, and not for olanzapine, quetiapine, or risperidone.

In FIG. 12, a sample containing olanzapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains olanzapine antibody (4G9-1) in reaction zone 4. A typicaldose response curve was generated as is shown in FIG. 12 only forolanzapine, and not for aripiprazole, quetiapine, or risperidone.

In FIG. 13, a sample containing quetiapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains quetiapine antibody (11) in reaction zone 6. A typicaldose response curve was generated as is shown in FIG. 13 only forquetiapine, and not for aripiprazole, olanzapine, or risperidone.

In FIG. 14, a sample containing risperidone is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains risperidone antibody (5-9) in reaction zone 8. A typicaldose response curve was generated as is shown in FIG. 14 only forrisperidone, and not for aripiprazole, olanzapine, or quetiapine.

FIGS. 15-18 show typical dose response curves for each assay in thepresence of other conjugates and antibodies. In FIG. 15, a samplecontaining aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (again containing labeledaripiprazole, labeled olanzapine, labeled quetiapine, and labeledrisperidone) and to the reaction zone. The reaction zone again containsaripiprazole antibody (5C7) in reaction zone 2, as well as olanzapineantibody (4G9-1) in reaction zone 4, quetiapine antibody (11) inreaction zone 6, and risperidone antibody (5-9) in reaction zone 8. Atypical dose response curve was generated for aripiprazole, as is shownin FIG. 15. When a sample containing olanzapine was deposited in thesample zone of this chip, a typical dose response curve was generatedfor olanzapine as shown in FIG. 16. When a sample containing quetiapinewas deposited in the sample zone of this chip, a typical dose responsecurve for quetiapine was generated as shown in FIG. 17. When a samplecontaining risperidone was deposited in the sample zone of this chip, atypical dose response curve for risperidone was generated as shown inFIG. 18.

FIGS. 19-22 show comparisons of dose response curves generated aspositive controls (FIGS. 7-10) to dose response curves generated in themultiplex format (FIGS. 15-18). The comparison for aripiprazole is shownin FIG. 19; for olanzapine in FIG. 20; for quetiapine in FIG. 21; andfor risperidone in FIG. 22. These figures show that the positive controlcurves are similar to the multiplex curves.

These data show that a lateral flow assay device of the subjectinvention can be used to detect multiple anti-psychotic drugs using asingle sample from a patient on one portable, point-of-care device.

What is claimed is:
 1. A method of detecting risperidone in a sample,the method comprising: (i) generating an antibody or a binding fragmentthereof that specifically binds to risperidone in response to aconjugate of a compound of Formula I and an immunogenic carrier, FormulaI:

wherein: R¹ is H, or OH; R² is

or O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H; wherein: r is 1, 2, 3, 4, or 5; m is1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or 5; (ii) contacting a samplewith the antibody or the binding fragment thereof labeled with adetectable marker, wherein the labeled antibody or the labeled bindingfragment and risperidone present in the sample form a labeled complex;and (iii) detecting the labeled complex thereby detecting risperidone inthe sample.
 2. The method of claim 1, wherein the detection ofrisperidone is an indication of patient adherence with prescribedrisperidone therapy.
 3. The method of claim 1, wherein the detection ofrisperidone is used to determine whether a patient should be convertedfrom an oral risperidone regimen to an injectable anti-psychoticregimen.
 4. The method of claim 1, wherein the detection of risperidoneis used to determine if the dose level or dosing interval of oral orinjectable risperidone should be increased or decreased to ensureattainment or maintenance of efficacious or safe drug levels.
 5. Themethod of claim 1, wherein the detection of risperidone is an aid in theinitiation of risperidone therapy by providing evidence of theattainment of minimum pK levels.
 6. The method of claim 1, wherein thedetection of risperidone is used to determine bioequivalence ofrisperidone in multiple formulations or from multiple sources.
 7. Themethod of claim 1, wherein the detection of risperidone is used toassess the impact of polypharmacy and potential drug-drug interactions.8. The method of claim 1, wherein the detection of risperidone is anindication that a patient should be excluded from or included into aclinical trial and is an aid in the subsequent monitoring of adherenceto clinical trial medication requirements.
 9. The method of claim 1,further comprising detecting the presence of one or more analytes inaddition to risperidone.
 10. The method of claim 9, wherein the one ormore analytes are anti-psychotic drugs other than risperidone.
 11. Themethod of claim 10, wherein the anti-psychotic drugs other thanrisperidone are selected from the group consisting of: paliperidone,aripiprazole, quetiapine, olanzapine, and metabolites thereof.
 12. Acompetitive immunoassay method for detecting sample risperidone in asample, the method comprising: (i) generating an antibody or a bindingfragment thereof that specifically binds to risperidone in response to aconjugate of a compound of Formula I and an immunogenic carrier, FormulaI:

wherein: R¹ is H, or OH; R² is

or O(CH₂)_(r)NHC(O)(CH₂)_(m)CO₂H; wherein: r is 1, 2, 3, 4, or 5; m is1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or 5; (ii) contacting a samplewith the antibody or the binding fragment thereof, and with risperidoneor a competitive binding partner of risperidone, wherein one of theantibody or the binding fragment thereof and the risperidone or thecompetitive binding partner thereof is labeled with a detectable marker,and wherein the sample risperidone competes with the risperidone or thecompetitive binding partner thereof for binding to the antibody or thebinding fragment thereof; and (ii) detecting the detectable markerthereby detecting the sample risperidone.
 13. The method of claim 12,wherein the risperidone or the competitive binding partner thereof islabeled with the detectable marker.
 14. The method of claim 12, whereinthe antibody or the binding fragment thereof is labeled with thedetectable marker.
 15. The method of claim 12, wherein the immunoassayis performed on a lateral flow assay device and the sample is applied tothe device.
 16. The method of claim 12, wherein the detection ofrisperidone is an indication of patient adherence with prescribedrisperidone therapy.
 17. The method of claim 12, wherein the detectionof risperidone is used to determine whether a patient should beconverted from an oral risperidone regimen to an injectableanti-psychotic regimen.
 18. The method of claim 12, wherein thedetection of risperidone is used to determine if the dose level ordosing interval of oral or injectable risperidone should be increased ordecreased to ensure attainment or maintenance of efficacious or safedrug levels.
 19. The method of claim 12, wherein the detection ofrisperidone is an aid in the initiation of paliperidone therapy byproviding evidence of the attainment of minimum pK levels.
 20. Themethod of claim 12, wherein the detection of risperidone is used todetermine bioequivalence of risperidone in multiple formulations or frommultiple sources.
 21. The method of claim 12, wherein the detection ofrisperidone is used to assess the impact of polypharmacy and potentialdrug-drug interactions.
 22. The method of claim 12, wherein thedetection of risperidone is an indication that a patient should beexcluded from or included into a clinical trial and is an aid in thesubsequent monitoring of adherence to clinical trial medicationrequirements.
 23. The method of claim 12, further comprising detectingthe presence of one or more analytes in addition to risperidone.
 24. Themethod of claim 23, wherein the one or more analytes are anti-psychoticdrugs other than risperidone.
 25. The method of claim 24, wherein theanti-psychotic drugs other than risperidone are selected from the groupconsisting of: paliperidone, aripiprazole, quetiapine, olanzapine, andmetabolites thereof.